Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies


Marius, P., Wright, J. N., Findlow, I.S. and Williamson, P.T.F. (2010) Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies Protein Expression and Purification, 72, (1), pp. 107-112. (doi:10.1016/j.pep.2010.01.019). (PMID:20117215).

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Description/Abstract

Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of ?-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi. To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be performed. Expressing the N-terminal domain of Fukutin-1 fused to a His6 tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4 mg per litre of minimal media. Preliminary biophysical analyses have confirmed the identity of the peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured ?-helix as predicted from the sequence.

Item Type: Article
Digital Object Identifier (DOI): doi:10.1016/j.pep.2010.01.019
ISSNs: 1046-5928 (print)
Keywords: expression, purification, transmembrane peptides, solid-state nmr
Subjects:
Organisations: Molecular and Cellular
ePrint ID: 335788
Date :
Date Event
July 2010Published
Date Deposited: 13 Mar 2012 13:36
Last Modified: 17 Apr 2017 17:25
Further Information:Google Scholar
URI: http://eprints.soton.ac.uk/id/eprint/335788

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